N-acetyl-d-glucosamine for enhanced specificity of strep a immunoassay

ABSTRACT

Methods, compositions and kits for detecting Group A  streptococcus  in a biological sample are described. More particularly, the present disclosure provides an immunoassay in which the specificity of detection of Group A  streptococcus  is enhanced by addition of N-acetyl-D-glucosamine. These methods, compositions and kits are useful in convenient, reliable and early diagnosis of streptococcal infection in a human subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/563,542, filed Jul. 31, 2012, now allowed; which claims the benefitof U.S. Application No. 61/514,790, filed Aug. 3, 2011, each isincorporated by reference herein in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to the field of diagnostics,and, in particular, to devices, methods and kits for detecting analytesin biological samples. More particularly, the present disclosureprovides an immunoassay in which the specificity of detection of Group Astreptococcus is enhanced by addition of N-acetyl-D-glucosamine.

BACKGROUND

Streptococcus pyogenes (Group A streptococcus) is a Gram-positive,nonmotile, non-sporeforming bacterium that occurs in chains or in pairsof cells, where individual cells are round-to-ovoid cocci, 0.6-1.0micrometer in diameter. The cell surface structure of Group Astreptococci is composed of repeating units of N-acetylglucosamine andN-acetylmuramic acid, the standard peptidoglycan. Historically, thedefinitive identification of streptococci has rested on the serologicreactivity of “cell wall” polysaccharide antigens as originallydescribed by Rebecca Lancefield. Eighteen group-specific antigens(Lancefield groups) were established. The Group A capsularpolysaccharide (also called “C substance” or “group carbohydrateantigen”) is a polymer of N-acetylglucosamine and rhamnose. Some groupantigens are shared by more than one species. (K. Todar, Online Textbookof Bacteriology; See textbookofbacteriology.net).

S. pyogenes is one of the most frequent pathogens of humans.Approximately 5-15% of normal individuals harbor the bacterium, usuallyin the respiratory tract, yet remain asymptomatic. As normal flora, S.pyogenes can infect when defenses are compromised or when the organismsare able to penetrate the constitutive defenses. When the bacteria areintroduced or transmitted to vulnerable tissues, a variety of types ofsuppurative infections can occur.

Acute diseases associated with S. pyogenes occur mainly in respiratorytract, bloodstream or skin. Streptococcal disease is most often arespiratory infection (pharyngitis or tonsillitis) or a skin infection(pyoderma). Acute S. pyogenes infections may present as pharyngitis(strep throat), scarlet fever (rash), impetigo (infection of thesuperficial layers of the skin) or cellulitis (infection of the deeplayers of the skin). Invasive, toxigenic infections can result innecrotizing fasciitis, joint or bone infections, myositis, meningitis,endocarditis and streptococcal toxic shock syndrome. Patients may alsodevelop immune-mediated post-streptococcal sequelae, such as acuterheumatic fever and acute glomerulonephritis, following acute infectionscaused by S. pyogenes, which occur in 1-3% of untreated infections.These conditions and their pathology are not attributable todissemination of bacteria, but to aberrant immunological reactions toGroup A streptococcal antigens.

Because penicillin is effective in treatment of Group A streptococcaldisease, the majority of infections amount to no more than pharyngitisaccompanied by a rash. However, due to the occasional cases of rapidlyprogressive disease and because of the small risk of serious sequelae inuntreated infections, S. pyogenes remains a major health concern, andeffort is being directed toward clarifying the risk and mechanisms ofthese sequelae and identifying rheumatogenic and nephritogenic strainsof streptococci.

The cell surface of S. pyogenes accounts for many of the bacterium'sdeterminants of virulence, especially those concerned with colonizationand evasion of phagocytosis and the host immune responses. The surfaceof the bacterium is incredibly complex and chemically-diverse. Antigeniccomponents include capsular polysaccharide (C-substance), cell wallpeptidoglycan and lipoteichoic acid (LTA), and a variety of surfaceproteins, including M protein, fimbrial proteins, fibronectin-bindingproteins, (e.g. Protein F) and cell-bound streptokinase.

The cytoplasmic membrane of S. pyogenes contains some antigens similarto those of human cardiac, skeletal, and smooth muscle, heart valvefibroblasts, and neuronal tissues. Molecular mimicry between pathogenand host has been proposed as a mechanism for the development ofautoimmune diseases. Because microorganisms contain proteins similar tohost proteins, the host's immune response may be suppressed or tolerantto infection. Conversely, stimulation of the host's B and T cells by amolecular mimic can cause the host's immune system to begin respondingto self proteins as if they are foreign.

As in other autoimmune diseases, both environmental and genetic factorsare involved in the development of rheumatic carditis and inflammatoryheart disease, and molecular mimicry between the group A streptococcusand heart tissues appears to play a role. The study of B and T cellresponses against group A streptococcal antigens has yielded someinformation about several steps in the pathogenesis of rheumaticcarditis following group A streptococcal infection. An early stepinvolves the development of crossreactive autoantibodies against thegroup A streptococcal carbohydrate antigen N-acetyl-glucosamine andcardiac myosin. These antibodies then react with valvular endothelium,which becomes inflamed with expression of vascular cell adhesionmolecule-1 (VCAM-1). T cells, CD4+ and CD8+, then infiltrate through theendothelium/endocardium into the valve (an avascular structure). Aschoffbodies or granulomatous lesions may form containing macrophages and Tcells underneath the endocardium. The T cells are responsive tostreptococcal M protein antigen sequences. The valve becomes scarredwith eventual neovascularization and progressive, chronic disease in thevalve. In the host, the mimicking antigens cardiac myosin and lamininhave been involved in the myocardium and valve, respectively.(Cunningham, Front. Biosci., 2003, 8:s533-43).

Rheumatic fever (RF) and the antiphospholipid syndrome (APS) areautoimmune diseases sharing similar cardiac and neurologicalpathologies. There appears to be a considerable overlap of humoralimmunity in RF and APS, supporting a hypothesis that common pathogenicmechanisms underlie the development of cardiac valve lesions and CentralNervous System abnormalities in both diseases. The pathogenic moleculesengaged in these autoimmune conditions, M protein,N-acetyl-beta-D-glucosamine (also called “NAG” or “GlcNAc”) and beta2glycoprotein-I (beta2GPI), were found to share some epitopes. Theimmunoglobulin G sera from APS patients contained a considerableanti-streptococcal M protein as well as anti-GlcNAc activity.Furthermore, beta2GPI inhibited anti-GlcNAc activity from APS patientswith chorea. (Blank, et al., 2006, Rheumatology (Oxford). 45(7):833-41).

Detection of microbial pathogens in biological samples is of particularvalue in clinical medicine, as treatment may vary considerably dependingupon the causative organism. Thus, the accurate and rapid identificationof pathogens in biological samples of patients suspected of having aninfectious disease can be critical to provide prompt and appropriatetreatment to patients. Rapid identification of disease-causing organismsin biological samples is important even for non-life threateninginfections.

Rapid methods of diagnosing microbial infections have been developed toprovide timely results for guiding clinical therapy. Some of the mosteffective of these rapid methods have been immunologically based.Monoclonal and polyclonal antibodies to microbe-specific antigens havebeen developed and used in immunoassays to identify specific microbes inbiological samples. For example, immunoassays for the identification ofgroup A streptococcal antigens in human samples are useful for the earlydetection of S. pyogenes infection, so that proper antibiotic treatmentmay be started.

Group A Streptococcus in pharyngeal exudates can be identified bypolyclonal antibodies to antigens specific for Group A streptococcus.One such test is described in U.S. Pat. No. 5,770,460, providing aone-step lateral flow assay for Group A streptococcus-specific antigens.However, tests relying on pharyngeal swabs are often complicated by ahigh false positive rate. Although instructions for use of pharyngealswab tests specifically direct the user to avoid contacting the tongue,cheek and/or teeth with the swab, inadvertent contact often occurs,nonetheless. Epithelial cells originating from the tongue, cheek and/orteeth may contain molecular mimics of one or more components of the S.pyogenes cell wall, and the polyclonal antibody specific for Group Astreptococcus may bind and “recognize” epitopes on the epithelial cellsin a test subject not infected by or carrying Group A strep, resultingin a false positive result. A highly specific and facile immunoassaywith a reduced rate of false positives is needed to provide accuratedetection of Group A streptococcus infection. Quite surprisingly, thepresent disclosure fulfills these and other related needs.

BRIEF SUMMARY

The present disclosure provides devices, methods and improved diagnostickits for detecting Group A streptococcus (also referred to herein as“Strep A”) in biological samples. More particularly, the presentdisclosure provides an improved immunoassay in which Group Astreptococcal infection is detected with a reduced rate offalse-positive results via addition of N-acetyl-D-glucosamine to theassay.

In one aspect, a device for detecting the presence of Group Astreptococcus in a sample is provided. The device comprises a matrixhaving (i) a sample receiving zone for receiving a sample containing orsuspected of containing a Group A streptococcus-specific antigen, (ii) alabeling zone containing an antibody for specifically labeling theantigen as it passes there through and (iii) a capture zone having meansfor specifically binding the labeled antigen thereon, wherein the samplereceiving zone, the labeling zone and the capture zone are arranged onthe matrix in a liquid flow path, and wherein at least one of the samplereceiving zone and the labeling zone comprise N-acetyl-D-glucosamine(NAG).

In one embodiment, the antibody is a polyclonal antibody.

In another embodiment, the antibody is fluorescently labeled.

In still another embodiment, the means for specifically binding thelabeled antigen is a capture antibody. In one embodiment, the captureantibody is a polyclonal antibody.

In another aspect, a kit is provided. The kit comprises a devicecomprising a matrix having (i) a sample receiving zone for receiving asample containing or suspected of containing a Group Astreptococcus-specific antigen, (ii) a labeling zone containing anantibody for specifically labeling the antigen as it passes therethrough and (iii) a capture zone having means for specifically bindingthe labeled antigen thereon, wherein the sample receiving zone, thelabeling zone and the capture zone are arranged on the matrix in aliquid flow path; and a container comprising an extraction reagent. Atleast one of the extraction reagent, the sample receiving zone and thelabeling zone comprise N-acetyl-D-glucosamine (NAG).

In one embodiment, NAG is deposited on the sample receiving zone.

In another aspect, a method for detecting the presence or absence ofGroup A streptococcus in a biological sample is provided. The methodcomprises providing a device or a kit as described herein placing abiological sample on the device; and determining the presence or absenceof Group A streptococcus, for example by visually reading (with theunaided eye, with an instrument, or with the eye assisted by aninstrument) the result on the test line of the device.

In one embodiment, the method further comprises providing an instrumentfor collecting the biological sample; and collecting a biological sampleon the instrument.

In still another embodiment, the method further comprises providinginstructions for use, wherein the instructions do not caution to nottouch the tongue, sides or top of mouth with the instrument whencollecting the sample.

In another aspect, a kit is provided. The kit comprises a deviceaccording to any of the embodiments described herein, a containercomprising an extraction reagent; and an instrument for collecting abiological sample; and instructions for use.

In one embodiment, the instrument is a swab.

In another embodiment, the instructions do not caution to not touch thetongue, sides or top of mouth with the instrument when collecting thesample.

In still another aspect, a method to reduce the false positive rate of alateral flow assay in the detection of Group A streptococcus in asample, wherein, in the lateral flow assay, N-acetyl-D-glucosamine(NAG)-binding components of a polyclonal antibody label used in theassay are preferentially bound is provided. The method comprise treatinga bibulous matrix with an amount of N-acetyl-D-glucosamine (NAG)effective as a blocking agent to enhance the specific binding of thepolyclonal antibody to Group A streptococcus antigen and reduce thefalse positive rate of the assay.

In one embodiment, a device for detecting the presence of Group Astreptococcus in a sample is provided. The device comprises a receivingchamber for receiving a sample suspected of comprising Group Astreptococcus-specific antigen, the chamber dimensioned to receive aliquid extraction reagent and N-acetyl-D-glucosamine (NAG) that combinewith the sample to form a treated sample; and a matrix having a samplereceiving zone for receiving the treated sample, a labeling zone havinga polyclonal antibody for specifically labeling the antigen as it passesthere through and a capture zone having means for specifically bindingthe labeled antigen thereon, wherein the sample receiving zone, thelabeling zone and the capture zone are arranged on the matrix in aliquid flow path.

In another embodiment, a method for detecting the presence of Group Astreptococcus in a sample is provided. The method comprises providing amatrix having (i) a sample receiving zone for receiving a samplecontaining or suspected of containing a Group A streptococcus-specificantigen, (ii) a labeling zone containing an antibody for specificallylabeling the antigen as it passes there through and (iii) a capture zonehaving means for specifically binding the labeled antigen thereon,wherein the sample receiving zone, the labeling zone and the capturezone are arranged on the matrix in a liquid flow path, and contactingthe sample receiving zone with the sample, wherein said sample istreated with a liquid reagent comprising N-acetyl-D-glucosamine (NAG)prior to contacting; and detecting the presence or absence of theantigen in the capture zone.

In another embodiment, a device for detecting the presence of Group Astreptococcus in a sample is provided. The device comprises a receivingchamber for receiving a sample suspected of comprising Group Astreptococcus-specific antigen, the chamber dimensioned to receive aliquid extraction reagent that contacts the sample to provide a treatedsample; and a matrix having a sample receiving zone for receiving thetreated sample, a labeling zone containing N-acetyl-D-glucosamine (NAG)and a polyclonal antibody for specifically labeling the antigen as itpasses there through and a capture zone having means for specificallybinding the labeled antigen thereon, wherein the sample receiving zone,the labeling zone and the capture zone are arranged on the matrix in aliquid flow path.

In yet another embodiment, a device for detecting the presence of GroupA streptococcus in a sample is provided. The device comprises areceiving chamber for receiving a sample suspected of comprising Group Astreptococcus-specific antigen, the chamber dimensioned to receive aliquid extraction reagent that contacts the sample to provide a treatedsample; and a matrix having a sample receiving zone for receiving thetreated sample, a labeling zone containing a polyclonal antibody forspecifically labeling the antigen as it passes there through and acapture zone containing N-acetyl-D-glucosamine (NAG) and a means forspecifically binding the labeled antigen thereon, wherein the samplereceiving zone, the labeling zone and the capture zone are arranged onthe matrix in a liquid flow path.

In still another embodiment, a method to reduce the false positive rateof a lateral flow assay in the detection of Group A streptococcus in asample is provided, where in the lateral flow assay,N-acetyl-D-glucosamine (NAG)-binding components of a polyclonal antibodylabel used in the assay are preferentially bound. The method comprisesadding to an extraction reagent, or to a localized region of theimmunoassay test strip an amount of N-acetyl-D-glucosamine (NAG)effective as a blocking agent to enhance the specific binding of thepolyclonal antibody to Group A streptococcus antigen and reduce thefalse positive rate of the assay.

In another embodiment, an improvement in an immunoassay device fordetecting Group A streptococcus having a housing with at least oneopening there through for introduction of a liquid sample into thehousing, an extraction reagent, a sample receiving zone of porousmaterial in said housing to be contacted by said liquid sample, and apolyclonal antibody labeling agent in a labeling zone is provided. Theimprovement comprises N-acetyl-D-glucosamine (NAG) added to the samplereceiving zone in an amount effective as a blocking agent to enhance thespecific binding of the polyclonal antibody to Group A streptococcusantigen and reduce the false positive rate of the assay.

In still another embodiment, an improvement in an immunoassay device fordetecting Group A streptococcus having a housing with at least oneopening therethrough for introduction of a liquid sample into thehousing, an extraction reagent, a sample receiving zone of porousmaterial in said housing to be contacted by said liquid sample, and apolyclonal antibody labeling agent in a labeling zone is provided. Theimprovement comprises N-acetyl-D-glucosamine (NAG) added to the labelingzone in an amount effective as a blocking agent to enhance the specificbinding of the polyclonal antibody to Group A streptococcus antigen andreduce the false positive rate of the assay.

In yet another embodiment, an improvement in an immunoassay device fordetecting Group A streptococcus having a housing with at least oneopening there through for introduction of a liquid sample into thehousing, an extraction reagent, a sample receiving zone of porousmaterial in said housing to be contacted by said liquid sample, and apolyclonal antibody labeling agent in a labeling zone is provided. Theimprovement comprises N-acetyl-D-glucosamine (NAG) added to theextraction reagent in an amount effective as a blocking agent to enhancethe specific binding of the polyclonal antibody to Group A streptococcusantigen and reduce the false positive rate of the assay.

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the absolute count of Strep A antigen inthroat swabs taken from healthy individuals (C-001 to C-030), innegative and positive control samples, each tested using a lateral flowdiagnostic immunoassay for Strep A;

FIG. 2 is a bar graph showing the absolute value of the signal at thetest line in Strep A immunoassay devices for samples collected from thesaliva (whole and fractions after centrifugation), cheek, tongue, nose,ear and skin of a human, and from the laboratory bench top surface;

FIG. 3 is a bar graph showing the absolute value of the signal at thetest line of a Strep A immunoassay device for saliva samples fromhealthy individuals (C-001-C-0030) where a known quantity of anti-groupA strep antibody was added to the extraction reagent when preparing thesamples for deposition on the immunoassay;

FIG. 4 is a bar graph showing the relative signal observed at the testline of an immunoassay device for detection of Strep A for tongue swabsamples prepared for testing on the device by including the indicatedchemical compound in the extraction reagent;

FIG. 5 illustrates an exemplary immunoassay device;

FIG. 6 is a graph of relative signal at the test line of an immunoassaydevice for Strep A as a function of concentration of NAG present in theextraction reagent for tongue swab samples (diamonds) and for a Strep Aquality control standard of 2×10³ organisms/test (squares);

FIG. 7 is a bar graph showing the data presented in FIG. 1 and thesample samples tested again on an immunoassay Strep A detection stripwith 0.5 mg/mL NAG added to the extraction reagent (right hand bar ineach of the data sets for each of the indicated patients);

FIG. 8 is a bar graph showing the absolute signal at the test line of aStrep A immunoassay device comprising NAG in the sample pad of thedevice by depositing, and allowing to dry, a solution to the sample padof NAG at concentrations of 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL and 1 mg/mLand testing for signal upon application of saline (negative control,left bar in each data set), a quality control standard of 2×10³organisms/test (middle bar in each data set) and a tongue swab (rightbar in each data set); and

FIG. 9 is a table showing the calculation of the sensitivity,specificity, positive and negative predictive values of an immunoassayfor Group A streptococcus improved with the addition of NAG, in accordwith the invention herein.

These and other embodiments are further described in the detaileddescription that follows.

DETAILED DESCRIPTION I. Definitions

Before the present methods and compositions are described, it is to beunderstood that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. Several embodiments of thepresent disclosure are described in detail hereinafter. Theseembodiments may take many different forms and should not be construed aslimited to those embodiments explicitly set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the present disclosure tothose skilled in the art. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe invention will be limited only by the appended claims.

All patents, applications, published applications and other publicationsreferred to herein are incorporated by reference in their entirety.

As used herein, the following terms are intended to have the followingmeanings:

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a protein” includes a plurality of such proteinsand reference to “the formulation” includes reference to one or moreformulations and equivalents thereof known to those skilled in the art,and so forth.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed bythis disclosure. The upper and lower limits of these smaller ranges mayindependently be included or excluded in the range, and each range whereeither, neither or both limits are included in the smaller ranges isalso encompassed by this disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also within the scope of this disclosure.

“Protein,” “polypeptide,” “oligopeptide,” and “peptide” are usedinterchangeably to denote a polymer of at least two amino acidscovalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

N-Acetylglucosamine (also called “N-acetyl-D-glucosamine,” “NAG” or“GlcNAc”) is a monosaccharide derivative of glucose having a molecularformula of C₈H₁₅NO₆, a molar mass of 221.21 g/mol. It is part of abiopolymer in bacterial cell walls, and, in particular, the cell surfacestructure of Streptococcus pyogenes (Group A streptococcus) comprisesalternating units of NAG and N-acetylmuramic acid (MurNAc), cross-linkedwith oligopeptides at the lactic acid residue of MurNAc. This layeredstructure is called peptidoglycan. NAG is the monomeric unit of thepolymer chitin, which forms the exoskeletons of insects and crustaceans.NAG polymerized with glucuronic acid forms hyaluronan, a component ofconnective, epithelial and neural tissues of higher organisms.

The term “sequence identity” means nucleic acid or amino acid sequenceidentity in two or more aligned sequences, aligned using a sequencealignment program.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at (ncbi.nlm.gov/BLAST/). See, also,Altschul, S. F. et al., 1990 and Altschul, S. F. et al., 1997.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage may be calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Alternatively, the percentage may be calculated by determiningthe number of positions at which either the identical nucleic acid baseor amino acid residue occurs in both sequences or a nucleic acid base oramino acid residue is aligned with a gap to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. Those of skill in theart appreciate that there are many established algorithms available toalign two sequences. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package),or by visual inspection (see generally, Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement) (Ausubel)). Examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

While all of the above mentioned algorithms and programs are suitablefor a determination of sequence alignment and % sequence identity, forpurposes of the disclosure herein, determination of % sequence identitywill typically be performed using the BESTFIT or GAP programs in the GCGWisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

The phrase “% sequence identity” refers to the level of nucleic acid oramino acid sequence identity between two or more aligned sequences, whenaligned using a sequence alignment program. For example, 70% homologymeans the same thing as 70% sequence identity determined by a definedalgorithm, and accordingly a homologue of a given sequence has greaterthan 70% sequence identity over a length of the given sequence.Exemplary levels of sequence identity include, but are not limited to70%, 75% 80%, 85%, 90% or 95%, 96%, 97%, 98% or 99% sequence identity toa given sequence, e.g., the nucleic acid or amino acid sequence of aprotein, as described herein.

“Associated” refers to coincidence with the development or manifestationof a disease, condition or phenotype. Association may be due to, but isnot limited to, genes responsible for housekeeping functions whosealteration can provide the foundation for a variety of diseases andconditions, those that are part of a pathway that is involved in aspecific disease, condition or phenotype and those that indirectlycontribute to the manifestation of a disease, condition or phenotype.

As pertains to the present disclosure, a biological fluid can be asolid, or semi-solid sample, including feces, biopsy specimens, skin,nails, and hair, or a liquid sample, such as urine, saliva, sputum,mucous, blood, blood components such as plasma or serum, amniotic fluid,semen, vaginal secretions, tears, spinal fluid, washings, and otherbodily fluids. Included among the sample are swab specimens from, e.g.,the cervix, urethra, nostril, and throat. Any of such samples may befrom a living, dead, or dying animal or a plant. Animals includemammals, such as humans.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies or fragment thereof made by recombinantmethods.

“Antibody” includes whole antibodies, including those of the IgG, IgMand IgA isotypes, and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chain thereof. An “antibody” refersto a glycoprotein comprising at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, or an antigen bindingportion thereof. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. TheIgG heavy chain constant region is comprised of four domains, CH1,hinge, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

“Isolated antibody,” as used herein, is intended to refer to an antibodywhich is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to the protein of interest is substantially free of antibodiesthat specifically bind antigens other than the protein of interest). Anisolated antibody that specifically binds to an epitope, isoform orvariant of the protein of interest may, however, have cross-reactivityto other related antigens, e.g., from other species (e.g., specieshomologs). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals. In some embodiments, acombination of “isolated” monoclonal antibodies having differentspecificities are combined in a well-defined composition.

“Specific binding” refers to antibody binding to a predeterminedantigen. Typically, the antibody binds with a dissociation constant (KD)of 10⁻⁷ M or less, and binds to the predetermined antigen with a KD thatis at least two-fold less than its KD for binding to a non-specificantigen (e.g., BSA, casein) other than the predetermined antigen or aclosely-related antigen. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

“Immunological binding,” as used herein, generally refers to thenon-covalent interactions of the type that occurs between an antibody,or fragment thereof, and the type 1 interferon or receptor for which theantibody is specific. The strength, or affinity, of immunologicalbinding interactions can be expressed in terms of the dissociationconstant (Kd) of the interaction, wherein a smaller Kd represents agreater affinity Immunological binding properties of selected antibodiescan be quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (Kon) and the “off rateconstant” (Koff) can be determined by calculation of the concentrationsand the actual rates of association and dissociation. The ratio ofKoff/Kon enables cancellation of all parameters not related to affinity,and is thus equal to the dissociation constant Kd. See, generally,Davies et al., Annual Rev. Biochem. 59:439-473 (1990).

“High affinity” for an IgG antibody refers to an antibody having a KD of10⁻⁸ M or less, more preferably 10⁻⁹ M or less and even more preferably10⁻¹⁰ M or less. However, “high affinity” binding can vary for otherantibody isotypes. For example, “high affinity” binding for an IgMisotype refers to an antibody having a KD of 10⁻⁷ M or less, morepreferably 10⁻⁸ M or less.

Monoclonal antibodies to a compound may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique originally described by Kohler & Milstein, 1975,Nature 256:495-497 and/or Kaprowski, U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique described by Kosbor et al., 1983, ImmunologyToday 4:72 and/or Cote et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030; and the EBV-hybridoma technique described by Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96. In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454; Boss, U.S. Pat. No. 4,816,397; Cabilly, U.S.Pat. No. 4,816,567) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Or“humanized” antibodies can be prepared (see, e.g., Queen, U.S. Pat. No.5,585,089). Alternatively, techniques described for the production ofsingle chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can beadapted to produce compound-specific single chain antibodies.

Antibody fragments which contain deletions of specific binding sites maybe generated by known techniques. For example, such fragments includebut are not limited to F(ab′)2 fragments, which can be produced bypepsin digestion of the antibody molecule and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for the peptide ofinterest.

The antibody or antibody fragment specific for the desired peptide canbe attached, for example, to agarose, and the antibody-agarose complexis used in immunochromatography to purify peptides. See, Scopes, 1984,Protein Purification: Principles and Practice, Springer-Verlag New York,Inc., N.Y., Livingstone, 1974, Methods In Enzymology: ImmunoaffinityChromatography of Proteins 34:723-731.

“Detect” and “detection” have their standard meaning, and are intendedto encompass detection, measurement and/or characterization of aselected protein or protein activity. For example, enzyme activity maybe “detected” in the course of detecting, screening for, orcharacterizing inhibitors, activators, and modulators of the protein.

The term “reference level” refers to a detected level of a positive ornegative control. For example, a reference level of a positive controlcan be a known amount of Group A streptococcus-specific antigen,obtained from a sample or culture of a known Group A streptococcusbacterium, a subject known to be infected with Group A streptococcus, orcan refer to a numerical value derived from known sources of Group Astreptococcus-specific antigen.

“Label” refers to any moiety that, when attached to a moiety describedherein, e.g., a peptide, protein or antibody, renders such a moietydetectable using known detection methods, e.g., spectroscopic,photochemical, electrochemiluminescent, and electrophoretic methods.Various labels suitable for use in the present disclosure include labelswhich produce a signal through either chemical or physical means,wherein the signal is detectable by visual or instrumental means.Exemplary labels include, but are not limited to, fluorophores andradioisotopes. Such labels allow direct detection of labeled compoundsby a suitable detector, e.g., a fluorometer. Such labels can includeenzymes and substrates, chromogens, catalysts, fluorescent compounds,phosphorescent compounds, chemiluminescent compounds, and radioactivelabels. Typically, a visually detectable label is used, therebyproviding for instrumental (e.g. spectrophotometer) readout of theamount of the analyte in the sample. Labels include enzymes such ashorseradish peroxidase, galactosidase (alpha and/or beta), and alkalinephosphatase. Suitable substrates include 3,3′,5,5′-tetramethylbenzidine(TMB) and 1,2 dioxetane. The method of detection will depend upon thelabeled used, and will be apparent to those of skill in the art.Examples of suitable direct labels include radiolabels, fluorophores,chromophores, chelating agents, particles, chemiluminescent agents andthe like.

For such embodiments, the label may be a direct label, i.e., a labelthat itself is detectable or produces a detectable signal, or it may bean indirect label, i.e., a label that is detectable or produces adetectable signal in the presence of another compound. “Labeled secondantibody” refers to an antibody that is attached to a detectable label.The label allows the antibody to produce a detectable signal that isrelated to the presence of analyte in the fluid sample.

Radioactive labels: Suitable radiolabels include, by way of example andnot limitation, ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ¹³¹I and ¹⁸⁶Re.

“Chromophore” refers to a moiety with absorption characteristics, i.e.,are capable of excitation upon irradiation by any of a variety ofphotonic sources. Chromophores can be fluorescing or non-fluorescing,and includes, among others, dyes, fluorophores, luminescent,chemiluminescent, and electrochemiluminescent molecules.

Examples of suitable indirect labels include enzymes capable of reactingwith or interacting with a substrate to produce a detectable signal(such as those used in ELISA and EMIT immunoassays), ligands capable ofbinding a labeled moiety, and the like. Suitable enzymes useful asindirect labels include, by way of example and not limitation, alkalinephosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphatedehydrogenase, lactate dehydrogenase and urease. The use of theseenzymes in ELISA and EMIT immunoassays is described in detail inEngvall, 1980, Methods Enzym. 70: 419-439 and U.S. Pat. No. 4,857,453.

“Substrate,” “support,” “solid support,” “solid varrier,” or “resin” areinterchangeable terms and refer to any solid phase material. Substratealso encompasses terms such as “solid phase,” “surface,” and/or“membrane.” A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. “Solid support” includes membranes (e.g. nitrocellulose),microtiter plate (e.g. PVC, polypropylene, polystyrene), dipstick, testtube, and glass or plastic beads. The configuration of a substrate canbe in the form of beads, spheres, particles, granules, a gel, a membraneor a surface. Surfaces can be planar, substantially planar, ornon-planar. Solid supports can be porous or non-porous, and can haveswelling or non-swelling characteristics. A solid support can beconfigured in the form of a well, depression, or other container,vessel, feature, or location. A plurality of supports can be configuredon an array at various locations, addressable for robotic delivery ofreagents, or by detection methods and/or instruments. Methods forimmobilizing biomolecules are well known in the art, and the antibodycan be attached covalently or non-covalently. In one embodiment, thesolid support is a stretavidin coated plate to which a biotinylatedantibody is non-covalently attached.

In statistics and diagnostic testing, sensitivity and specificity arestatistical measures of the performance of a binary classification test.Sensitivity (also called “recall rate”) measures the proportion ofactual positives which are correctly identified as such (e.g. thepercentage of sick people who are correctly identified as having thecondition). Specificity measures the proportion of negatives which arecorrectly identified (e.g. the percentage of healthy people who arecorrectly identified as not having the condition). These two measuresare closely related to the concepts of type I and type II errors. Atheoretical, optimal prediction aims to achieve 100% sensitivity (i.e.predict all people from the sick group as sick) and 100% specificity(i.e. not predict anyone from the healthy group as sick), howevertheoretically any predictor will possess a minimum error bound known asthe Bayes error rate.

“Specificity” relates to the ability of the diagnostic test to identifynegative results.

${Specificity} = \frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Negatives}}{\left( {{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Negatives}} + {\# \mspace{14mu} {of}\mspace{14mu} {False}\mspace{14mu} {Positives}}} \right)}$

If a test has high specificity, a positive result from the test means ahigh probability of the presence of the disease for which the test istesting.

“Sensitivity” relates to the ability of the diagnostic test to identifypositive results.

${Sensitivity} = \frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Positives}}{\left( {{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Positives}} + {\# \mspace{14mu} {of}\mspace{14mu} {False}\mspace{14mu} {Negatives}}} \right)}$

If a test has high sensitivity then a negative result would suggest theabsence of disease. For example, a sensitivity of 100% means that thetest recognizes all actual positives—i.e. all sick people are recognizedas being ill. Thus, in contrast to a high specificity test, negativeresults in a high sensitivity test are used to rule out the disease.

For any test, there is usually a trade-off between the measures. Forexample: in an airport security setting in which one is testing forpotential threats to safety, scanners may be set to trigger on low-riskitems like belt buckles and keys (low specificity), in order to reducethe risk of missing objects that do pose a threat to the aircraft andthose aboard (high sensitivity). This trade-off can be representedgraphically using a receiver operating characteristic (ROC) curve.

In some embodiments, a ROC is used to generate a summary statistic. Somecommon versions are: the intercept of the ROC curve with the line at 90degrees to the no-discrimination line (also called Youden's Jstatistic); the area between the ROC curve and the no-discriminationline; the area under the ROC curve, or “AUC” (“Area Under Curve”), or A′(pronounced “a-prime”); d′ (pronounced “d-prime”), the distance betweenthe mean of the distribution of activity in the system under noise-aloneconditions and its distribution under signal-alone conditions, dividedby their standard deviation, under the assumption that both thesedistributions are normal with the same standard deviation. Under theseassumptions, it can be proved that the shape of the ROC depends only ond′.

The “positive predictive value (PPV),” or “precision rate” of a test isa summary statistic used to describe the proportion of subjects withpositive test results who are correctly diagnosed. It is a measure ofthe performance of a diagnostic method, as it reflects the probabilitythat a positive test reflects the underlying condition being tested for.Its value does however depend on the prevalence of the outcome ofinterest, which may be unknown for a particular target population. ThePPV can be derived using Bayes' theorem.

The PPV is defined as:

${PPV} = {\frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Positives}}{\left( {{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Positives}} + {\# \mspace{14mu} {of}\mspace{14mu} {False}\mspace{14mu} {Positives}}} \right)} = \frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Positives}}{\# \mspace{14mu} {of}\mspace{14mu} {Positive}\mspace{14mu} {calls}}}$

where a “true positive” is the event that the test makes a positiveprediction, and the subject has a positive result under the goldstandard, and a “false positive” is the event that the test makes apositive prediction, and the subject has a positive result under thegold standard.

“Negative predictive value (NPV)” is defined as the proportion ofsubjects with a negative test result who are correctly diagnosed. A highNPV means that when the test yields a negative result, it is uncommonthat the result should have been positive. In the familiar context ofmedical testing, a high NPV means that the test only rarelymisclassifies a sick person as being healthy. Note that this saysnothing about the tendency of the test to mistakenly classify a healthyperson as being sick.

The NPV is determined as:

${NPV} = {\frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Negatives}}{\left( {{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Negatives}} + {\# \mspace{14mu} {of}\mspace{14mu} {False}\mspace{14mu} {Negatives}}} \right)} = \frac{\# \mspace{14mu} {of}\mspace{14mu} {True}\mspace{14mu} {Negatives}}{\# \mspace{14mu} {of}\mspace{14mu} {Negative}\mspace{14mu} {calls}}}$

where a “true negative” is the event that the test makes a negativeprediction, and the subject has a negative result under the goldstandard, and a “false negative” is the event that the test makes anegative prediction, and the subject has a positive result under thegold standard.

If the prevalence, sensitivity, and specificity are known, the positiveand negative predictive values (PPV and NPV) can be calculated for anyprevalence as follows:

${PPV} = \frac{{sensitivity} \times {prevalence}}{{{sensitivity} \times {prevalence}} + {\left( {1\text{-}{specificity}} \right) \times \left( {1\text{-}{prevalence}} \right)}}$${NPV} = \frac{{sensitivity} \times \left( {1\text{-}{prevalence}} \right)}{{\left( {1\text{-}{sensitivity}} \right) \times {prevalence}} + {{specificity} \times \left( {1\text{-}{prevalence}} \right)}}$

If the prevalence of the disease is very low, the positive predictivevalue will not be close to 1 even if both the sensitivity andspecificity are high. Thus in screening the general population it isinevitable that many people with positive test results will be falsepositives.

The rarer the abnormality, the more sure one can be that a negative testindicates no abnormality, and the less sure that a positive resultreally indicates an abnormality. The prevalence can be interpreted asthe probability before the test is carried out that the subject has thedisease, known as the prior probability of disease. The positive andnegative predictive values are the revised estimates of the sameprobability for those subjects who are positive and negative on thetest, and are known as posterior probabilities. The difference betweenthe prior and posterior probabilities is one way of assessing theusefulness of the test.

For any test result we can compare the probability of getting thatresult if the patient truly had the condition of interest with thecorresponding probability if he or she were healthy. The ratio of theseprobabilities is called the likelihood ratio, calculated assensitivity/(1-specificity). (Altman D G, Bland J M (1994). “Diagnostictests 2: Predictive values”, BMJ 309 (6947):102).

“Rule-out criteria” “Rule-Out,” or “RO” are terms used in a medicaldifferential diagnosis of a disease or condition, in which certaincriteria are evaluated in a clinical decision-making process ofelimination or inclusion. A subject is “ruled-out” when, uponconsideration of the criteria, the subject has been determined not tohave met all or a significant number of criteria for having a disease.

II. Devices and Kits Comprising NAG and Methods of Use

In a study conducted in support of the invention, throat swabs werecollected from thirty (30) healthy volunteers. Each sample was screenedfor the presence or absence of Strep A antigen using an immunoassay teststrip for detection of Strep A. Unexpectedly, a majority of these swabsgave a positive result, as seen from the data presented in FIG. 1 foreach person, denoted by the indicators C-001-0030. The negative controlsamples are shown as the three bars on the left and indicated“Noswab-Neg”, “eswab-Neg” and “eswab-Nebl.5”. The positive controlsample is on the far right, and denoted “Positive (2×10⁻³). At leastpatients C-001, C-002, C-007, C010, C-013, C-017, C-018, C-020, C-023,C-024 and C-030 were positive for Strep A. It was suspected that amajority of these were false positives because the samples were fromhealthy individuals. Further investigation was conducted to understandthe large number of false positives observed.

Package inserts of several commercial Strep A lateral flow tests, suchas QUICK-VUE® Strep A (Quidel Corporation), caution the clinician orperson collecting the sample on the swab to avoid touching the swab totongue, tonsils, cheek or teeth. Such caution was taken in the samplesobtained and tested in the study described above, so it was unclear whya large false positive rate was observed. Human biological materialsfrom the tongue, cheek and teeth in a subject's mouth were suspected asa likely cause of false positives using Strep A immunoassays.

Another study was conducted wherein a swab was used to collect samplesfrom a healthy human of saliva, inner cheek, tongue, nasal passagemucus, ear and skin. As a negative control, a swab of the laboratorybench surface was taken. The swabs were tested for the presence of StrepA using a lateral flow immunoassay. In this study, a sample of salivawas collected and centrifuged to obtain a supernatant and a pellet thatwere also tested. The results are shown in FIG. 2, and indicate that thesamples from the ear, skin and bench surface do not contain Strep Aantigen, as these samples have an absolute signal approximately the sameas the negative control samples (extraction reagent only, neat swab,PBS/BSA sample). However, the samples obtained from the mouth tissues,saliva, cheek and tongue, were positive for the presence of Strep A,relative to the negative controls.

Another study is described in Example 1, wherein saliva samples weretested for the presence or absence of Strep A with and without theaddition of a known amount of polyclonal anti-Strep A antibodies to thesamples. The results are shown in FIG. 3, where the bar on the left sideof each label along the x-axis corresponds to the saliva sample and thebar on the right side of each sample label on the x-axis corresponds tothe saliva sample plus addition of anti-Strep A antibodies. The threesamples on the far right correspond to quality control standards forStrep A with a known number of organisms with and without addition ofanti-strep A antibody. The data in the figure indicates that a componentin the saliva samples was causing the false positive readings at thetest line in the immunoassay device, rather than the Strep A antigens.

A further study was conducted to identify a possible source or cause ofthe false positive readings. In the study, detailed in Example 2, aseries of chemicals was tested for their ability to inhibit or block thebinding of the anti-Strep A polyclonal antibodies in the Strep Aimmunoassay. The compounds tested included N-acetyl glucosamine (NAG),glucosamine, acetyl-galactosamine, galactosamine, mannosamine,acetyl-muramic acid, chitin, chitosan and hyaluronic acid (HA-50K).Tongue swab samples from a healthy human patient and a Strep A qualitycontrol standard (1×10³ organisms/test) were obtained for testing. Usingan immunoassay device for detection of Strep A, the tongue swab sample(right hand bar for each data set in FIG. 4) and the QC standard (lefthand bar for each data set in FIG. 4) were each treated with theextraction reagents (such as the extraction reagents in a commerciallyavailable QUICK-VUE® Strep A kit) in the presence of the test compoundindicated along the x-axis in FIG. 4. As controls and comparators, theStrep A QC standard and a tongue swab sample were each also treated withthe extraction reagents (left data set in FIG. 4 labeled “Extr A+B” inFIG. 4; left bar is the positive control QC standard and the right barof the set of the tongue swab). Results are shown in FIG. 4, where therelative activity of the signal at the test line is shown with respectto its appropriate control. Of the compounds tested,N-acetyl-D-glucosamine (NAG) suppressed the signal resulting from thestrep A QC standard at the test line of the immunoassay device by morethan 50%, and was able to suppress the signal from the tongue swab to16% of the control tongue swab signal. The fact that false positivesignals from tongue swab specimens were efficiently suppressed byaddition of NAG suggests that the Strep A polyclonal antibody containssome non-specific binding activities to human tissue or cells from thetongue/mouth.

Accordingly, in one embodiment, the addition of NAG to an immunoassay iscontemplated, where the presence of NAG is effective to reducenon-specific binding between the antibodies in the Strep A polyclonalantibody population and components in the test sample by at least about50%, more preferably by at least about 60%, 70% or 75% of the signalobtained in the absence of NAG.

Without being bound to theory, the data suggests that the false positivesignals from glycoproteins present in epithelial cells inadvertentlycollected on pharyngeal swab specimens can be efficiently blocked orsuppressed by addition of NAG to the immunoassay. The polyclonalanti-Strep A antibodies used in the immunoassay may, in someembodiments, include a population of antibodies that recognize humanepithelial cell wall glycoproteins that mimic Group A streptococcal cellwall proteins. By providing a reagent to the immunoassay that can blockthis population of antibodies, the performance of the immunoassay interms of overall accuracy by reducing the rate of false positives can beimproved. The present invention meets this need by including NAG in theimmunoassay device and/or including NAG in extraction reagent(s)provided with the immunoassay.

Immunoassays for detection of Group A streptococcus that comprise NAGare contemplated, wherein the assay comprises a lateral flow device thatallows for one-step pretreatment and detection of Group A streptococcusorganisms with enhanced specificity. Immunoassay devices are known inthe art, and typically have at least a sample receiving zone, a labelingzone and a capture zone, and can be prepared according to thedescription in any of U.S. Pat. Nos. 5,415,994; 5,763,262 and 5,770,460,which are incorporated by reference in their entirety.

Accordingly, in one aspect of the disclosure, a device is provided fordetecting the presence of Group A streptococcus in a sample. Variousembodiments of a device are contemplated, and exemplary embodiments aredescribed herein for the purposes of illustration. A skilled artisanwill appreciate, however, that the illustrative embodiments arenon-limiting to the inventive concepts set forth herein.

In a general embodiment, a device comprises a series of zones in fluidcommunication. In a preferred embodiment, a sample receiving zone is influid communication with second and subsequent zones, such as a labelingzone, a capture zone, and/or an absorption zone. A first embodiment of adevice is depicted in FIG. 5, which shows an immunoassay test strip fordetection of Group A streptococcus. An exemplary test strip 10 iscomprised of a support layer 12 that preferably extends the length ofthe test strip. Support layer 12 supports in series a sample pad 14, alabel pad 16, a nitrocellulose member 17, and an optional absorbent pad18. On the nitrocellulose member is a test line 20 and a control line22. For detection of Strep A, the label pad comprises anti-Strep Aantibodies, as does the test line. In one embodiment, the antibodiesdeposited on the label pad comprise a label which aids or permitsdetection of the antibody. The labeled antibody specifically binds theStrep A antigen as it passes through the label zone. The capture zonecomprises a means for specifically binding the labeled antigen thereon.In accord with the present invention, the sample is contacted withN-acetyl-D-glucosamine (NAG) prior to application to the device and/orduring its flow through the device. Studies described herein illustratethe improved performance of a device intended for detecting Strep A whenNAG is incorporated in the assay. NAG can be incorporated into thedevice, such as in the sample receiving zone, the labeling zone, orboth, and/or the sample can be treated with NAG prior to its applicationto the sample receiving zone of device.

In one embodiment, the lateral flow immunoassay comprises an immunoassaywith label that can be read visually with the unaided eye, such as acolored bead or particle, wherein a collection of such beads orparticles at the test line of the immunoassay can be viewed by a userwith the naked eye. In another embodiment, the lateral flow immunoassaycomprises an immunoassay with a label that is read by an instrument orby an eye with the aid of an instrument. For example, a fluorescentlabel in the immunoassay is detected using an instrument that can excitethe label and the excited label can be read with the instrument, withthe eye aided by instrument or with the eye. An exemplary instrument andlateral flow immunoassay is described in U.S. Application No.61/666,689, which is incorporated by reference herein.

In another aspect, a device is provided for detecting the presence ofGroup A streptococcus in a sample, wherein the device comprises a matrixhaving (i) a sample receiving zone for receiving a sample containing orsuspected of containing a Group A streptococcus-specific antigen, (ii) alabeling zone containing an antibody for specifically labeling theantigen as it passes there through and (iii) a capture zone having meansfor specifically binding the labeled antigen thereon, wherein the samplereceiving zone, the labeling zone and the capture zone are arranged onthe matrix in a liquid flow path, and wherein the sample receiving zone,the labeling zone, or both contain NAG.

Another embodiment of a device contemplated for use is described in U.S.Pat. No. 5,415,994, which is incorporated by reference herein. In thisembodiment, the device comprises a receiving chamber positioned orpositionable for fluid contact with a lateral flow immunoassay device,and preferably positioned for fluid communication with a samplereceiving zone or a labeling zone of the immunoassay test strip. Thebiological sample suspect of containing Strep A is received into thereceiving chamber, such as by insertion of a swab containing the sampleor by dispensing an aliquot of the sample into the receiving chamber.One or more extraction or treatment agents can be additionally added tothe receiving chamber or to the swab. In one embodiment, the treatmentagent comprises NAG. In one embodiment, the receiving chamber ispositioned over the sample receiving zone is dimensioned for receiving aliquid extraction reagent comprising NAG, and, optionally comprises acylindrical portion for receiving a swab containing a patient sample.The immunoassay test strip comprises a matrix having a sample receivingzone for receiving the extraction liquid containing the treated samplesuspected of comprising Strep A antigen, a labeling zone having apolyclonal antibody for specifically labeling the antigen as it passesthere through and a capture zone having means for specifically bindingthe labeled antigen thereon, wherein the sample receiving zone, thelabeling zone and the capture zone are arranged on the matrix in aliquid flow path.

In some embodiments of the devices described herein, the extractionreagent provided to treat the biological sample contains NAG. In someembodiments, the labeling zone of the immunoassay device contains NAG.In some embodiments, the capture zone of the immunoassay device containsNAG. In some embodiments, the sample receiving zone of the immunoassaydevice contains NAG. In some embodiments, all or some of these specifiedzone comprises NAG, optionally in combination with NAG in an extractionreagent.

Devices as described above were used to further evaluation the effect ofNAG on device sensitivity and accuracy. In another study, described inExample 3, different concentrations of NAG in an immunoassay wereevaluated for inhibition of Strep A false positive signals. Tongue swabswere taken from a healthy individual, and a Strep A quality control (QC)standard (2.0×10³ organisms/test) were prepared for testing byextracting the sample using the provided extraction reagents, exceptthat NAG was added to the extraction reagents at 0.125 mg/mL, 0.25mg/mL, 0.5 mg/mL, 1 mg/mL and 2 mg/mL. The samples were deposited on theimmunoassay device. Results are shown in FIG. 6 as a function ofconcentration of NAG present in the extraction reagent for tongue swabsamples (diamonds) and for a Strep A quality control standard of 2×10³organisms/test (squares).

The results in FIG. 6 suggest that NAG more efficiently inhibited thefalse positives than suppressed Strep A positive signals. NAG present inthe immunoassay at a concentration of 0.5 mg/mL (volume of theextraction reagent) was able to inhibit false positives to approximately20% of the signal observed in the absence of NAG, while at the same NAGconcentration the positive signal of Strep A QC standard at 2.0×10³organisms/test remained at 50% of the signal observed in the absence ofNAG.

In another study, described in Example 4, the 30 samples originallycollected (FIG. 1) were tested again, this time with the addition of 0.5mg/mL NAG to the extraction reagent provided with the immunoassay testkit. FIG. 7 is a bar graph the presents again the data of FIG. 1 (leftbar of each data set) and an aliquot of the saliva sample from the samepatient (e.g., C-002) prepared with an extraction reagent comprising 0.5mg/mL NAG (right hand bar in each of the data sets for each of theindicated patients).

FIG. 8 shows the results of another study where patient samples weretested on an immunoassay device comprising NAG on the sample pad of thedevice. The devices were prepared by depositing to the sample pad, andallowing to dry, a solution of NAG at concentrations of 0.4 mg/mL, 0.6mg/mL, 0.8 mg/mL and 1 mg/mL. Tongue swab samples and Strep A QCstandards, along with a saline negative control, were each tested on adevice. In the bar graph of FIG. 8, the absolute signal at the test lineof a Strep A immunoassay device comprising NAG in the sample pad uponapplication of saline (negative control) is shown in the left bar ofeach data set, the quality control standard of 2×10³ organisms/test isshown in the middle bar of each data set and the tongue swab is shown inthe right bar of each data set. Inhibition of the positive signal wasobserved in the presence of NAG when the Strep A QC standard was tested,as well as for the tongue swab specimens. This result is consistent withthe results from study using NAG in the extraction solutions (FIG. 7 andExample 4, above). Accordingly, it is demonstrated that NAG can beincluded in an appropriate place on the immunoassay device itself or inan off-line extraction medium.

The data presented herein shows that addition of NAG to an immunoassayfor Strep A improves the specificity of the test for Group Astreptococcus, and reduces the rate of false positives. Thus, NAG can beused to effectively reduce false positives in such tests. A skilledartisan will appreciate that the amount of antibody, NAG and sample canbe adjusted for optimization of observing a positive signal from S.pyogenes while blocking false positives by NAG. The sensitivity andspecificity of the tests based on the studies conducted herein are shownin FIG. 9, which shows a calculation of the sensitivity, specificity,positive and negative predictive values of an improved Group Astreptococcus immunoassay.

Based on the foregoing, and in another aspect, a method is provided fordetecting the presence or absence of Strep A in a sample, comprising (a)providing a matrix having (i) a sample receiving zone for receiving asample containing or suspected of containing a Group Astreptococcus-specific antigen, (ii) a labeling zone containing anantibody for specifically labeling the antigen as it passes therethrough and (iii) a capture zone having means for specifically bindingthe labeled antigen thereon, wherein the sample receiving zone, thelabeling zone and the capture zone are arranged on the matrix in aliquid flow path, and (b) contacting the sample receiving zone with thesample, wherein said sample is treated with a liquid reagent comprisingNAG prior to contacting; and (c) detecting the presence or absence ofthe antigen in the capture zone. In one embodiment, the matrixadditionally comprises an absorbent zone downstream of the capture zone.

In another aspect, a method is provided to reduce the false positiverate of a lateral flow assay in the detection of Group A streptococcusin a liquid sample, wherein, in the lateral flow assay, NAG-bindingcomponents of a polyclonal antibody label used in the assay arepreferentially bound, the method comprising treating a bibulous matrixwith an amount of NAG effective as a blocking agent to enhance thespecific binding of the polyclonal antibody to Strep A antigen andreduce the false positive rate of the assay.

In another aspect, a method is provided to reduce the false positiverate of a lateral flow assay in the detection of Group A streptococcusin a liquid sample, wherein, in the lateral flow assay, NAG-bindingcomponents of a polyclonal antibody label used in the assay arepreferentially bound, the method comprising adding to an extractionreagent an amount of NAG effective as a blocking agent to enhance thespecific binding of the polyclonal antibody to Strep A antigen andreduce the false positive rate of the assay.

Improvements to a known immunoassay device for detecting Group Astreptococcus can be made by adding NAG to the sample receiving zone inan amount effective as a blocking agent to enhance the specific bindingof at least a portion of the polyclonal antibodies to Strep A antigen,to thereby reduce the false positive rate of the assay. Improvements canalso comprise adding NAG to the labeling zone in an amount effective asa blocking agent to enhance the specific binding of the polyclonalantibody to Strep A antigen and reduce the false positive rate of theassay. Improvements can also comprise adding NAG to the extractionreagent in an amount effective as a blocking agent to enhance thespecific binding of the polyclonal antibody to Strep A antigen andreduce the false positive rate of the assay.

In some embodiments, the sample is collected through the use of apharyngeal swab. In some embodiments, the sample is collected through aswab of the pharynx, tongue, cheek, teeth, gums or nasal passages. Insome embodiments, a body fluid is sampled, such as urine, saliva,sputum, mucous, blood, blood components such as plasma or serum,amniotic fluid, semen, wound secretions, vaginal secretions, tears,spinal fluid, washings, and other bodily fluids. Included among thesample are swab specimens from, e.g., the cervix, urethra, nostril, andthroat.

In some embodiments, the first antibody is a polyclonal antibody thatbinds to one or more epitopes of Group A streptococcus, and also bindsto NAG. In some embodiments, the antibody is a population of polyclonalantibodies, the population including a portion of antibodies havingspecific binding to NAG. In some embodiments, the antibody does not bindto glucosamine, galactosamine, mannosamine, acetyl-muramic acid, chitin,chitosan, and/or hyaluronic acid (e.g., HA-50K).

In some embodiments, the antibody has high specificity and lowsensitivity for detecting a Group A streptococcus antigen. In someembodiments, the antibody has high sensitivity and low specificity fordetecting a Group A streptococcus antigen. In some embodiments, theantibody has high specificity and high sensitivity for detecting a GroupA streptococcus antigen.

Examples of the antibodies used in the immunoassay of the presentdisclosure may include, but are not limited to a polyclonal antibody,such as an affinity purified rabbit anti-Strep A antibody.

Illustrative publications describing components of precursorcompositions, methods and kits, as well as various antibodies fordetecting Group A streptococcus include the following: U.S. Pat. Nos.5,415,994; 5,763,262 and 5,770,460. All of these patents, applicationsand publications are incorporated by reference herein, in theirentirety.

Kits

Kits comprising an immunoassay device as described herein are alsocontemplated. In addition to the immunoassay device, the kits mayadditionally include any one or more of written instructions for usingthe device and collecting a biological sample, an instrument or tool forcollecting a biological sample, labels for marking the device, acontainer or vial containing a reagent for preparing a treated sample.The kits may additionally include instructions for reading andinterpreting the results of an assay. The kits may further comprisereference samples that may be used to compare test results with thespecimen samples. In one embodiment the kits include a swab forcollecting a biological sample, and instructions for use of the assayand for collecting the sample, wherein the instructions do not contain acaution against contacting, for example, one or more of the back of thethroat, tonsils, cheek or tongue.

Accordingly, in another aspect, a kit is provided, comprising (a) adevice comprising a matrix having (i) a sample receiving zone forreceiving a sample containing or suspected of containing a Group Astreptococcus-specific antigen, (ii) a labeling zone containing anantibody for specifically labeling the antigen as it passes therethroughand (iii) a capture zone having means for specifically binding thelabeled antigen thereon, and, optionally (iv) an absorbent zone, whereinthe sample receiving zone, the labeling zone and the capture zone (andthe optional absorbent if present) are arranged on the matrix in aliquid flow path; and (b) a container comprising an extraction reagent;wherein at least one of the extraction reagent, the sample receivingzone and the labeling zone contain NAG.

Example 6 summarizes a study conducted using a kit comprised of alateral flow immunoassay test strip housed in a cassette with a samplereceiving chamber, a vial with a reagent solution and a dropper tip thatfit securely on the open end of the vial, a sterile rayon swab, apositive control swab that was coated with heat-inactivated,non-infectious Group A Streptoccus, a negative control swab that wascoated with heat-inactivated, non-infectious Group C Streptoccus, andinstructions for use. The test kit was used in a clinical study wheretwo throat swabs were obtained from 533 patients with symptomssuggestive of bacterial pharyngitis.

The lateral flow immunoassay test strip used in the study of Examplecontained NAG in dried form on the sample pad of the test strip (seeFIG. 5). The test strip was designed to work in conjunction with aninstrument capable or reading a fluorescent or luminescent signalemitted from the test line and the control line on the test strip. Theresults of the study are detailed in Example 6, where it can be seenthat the sensitivity and specificity were 99% and 96%, respectively.

EXAMPLES

The following examples describe exemplary assays that can be performedusing the presently disclosed methods and compositions. However, thepresent disclosure shall in no way be considered to be limited to theparticular embodiments described below.

Example 1: Study Using Saliva Samples from Healthy Volunteers

Saliva specimens were collected from thirty (30) healthy donors. As apositive control a quality control (QC) standard for Strep A consistingof a known number of antigens was obtained. QC standards with 3.75×10²organisms/test, 3.75×10⁴ organisms/test and 3.75×10⁵ organisms/test wereobtained. An aliquot of each saliva specimen was deposited on a lateralflow immunoassay test strip comprising similar to that shown in FIG. 5and comprising polyclonal antibodies specific for Strep A antigen with afluorescent label. A second aliquot of each saliva specimen was combinedwith a known quantity (2 mg/mL) of affinity purified polyclonal Strep Aantibody. The saliva plus antibody samples were deposited on theimmunoassay device, and the test line of the immunoassay device wasvisually observed for the presence of a fluorescent signal indicative ofantigen. The results are shown in FIG. 3.

As seen in FIG. 3, some of the saliva specimens indicated a positivesignal at the test line of the immunoassay device, when read using afluorescent analyzer. Anti-Strep A antibody added to the extraction mixwhen treating the sample inhibited the positive signal, as seen by theright hand bar for each sample in FIG. 3. Presence of the anti-Strep Aantibody in the saliva samples and the QC Standards attenuated thepositive signals differently. The attenuation in the presence of Strep Aantibody in the saliva specimens was less than on Strep A QC Standards.This can be seen by taking the ratios of the signals in the presence andabsence of the antibodies, as shown in Table 1, below. These resultsindicate that a component in the saliva specimens other than Strep Aantigen resulted in false positive signal.

TABLE 1 Saliva ID #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16#17 Saliva/ 3.2 4.1 1.3 4.0 1.3 1.0 3.0 1.7 5.5 4.7 2.7 2.8 2.6 5.4 5.05.1 5.1 Saliva + Ab QC QC QC Saliva ID Standard Standard Standard Org/#18 #19 #20 #21 #22 #23 #24 #25 #26 #27 #28 #29 #30 3.75 × 10² 3.75 ×10⁴ 3.75 × 10⁵ test Saliva/ 0.8 1.2 1.9 3.8 1.9 4.7 4.6 2.8 5.5 3.3 18.46.0 5.4 2.1 27.5 51.2 Saliva + Ab

Example 1: Analysis of Saccharide Compounds on Observed False PositiveResults

Compounds composed of or having structures similar to those in bacterialcell wall and human tissues were identified and selected forinvestigation for ability to inhibit generation of a positive signal ona strep A immunoassay test. The chemicals selected for study wereN-acetyl-D-glucosamine (NAG), glucosamine, acetyl-galactosamine,galactosamine, mannosamine, acetyl-muramic acid, chitin, chitosan andhyaluronic acid (HA-50K).

In the study, tongue swab samples from a healthy human patient wereobtained. A Strep A quality control standard (1×10³ organisms/test) wasalso obtained for testing. The tongue swab sample and the QC standardsample were each treated with one of the test compounds and the samplewas then deposited on an immunoassay device for detection of Strep A. Ascontrols and comparators, the Strep A QC standard and a tongue swab wereeach treated with the extraction reagents provided with the Strep Aimmunoassay test kit (left data set in FIG. 4 labeled “Extr A+B” in FIG.4; left bar is the positive control QC standard and the right bar of theset of the tongue swab). Results are shown in FIG. 4, where the relativeactivity of the signal at the test line is shown with respect to itsappropriate control for the tongue swab sample (right hand bar for eachdata set in FIG. 4) and the QC standard sample (left hand bar for eachdata set in FIG. 4). Of the compounds tested, N-acetyl-D-glucosamine(NAG) suppressed the signal resulting from the Strep A QC standard atthe test line of the immunoassay device by more than 50%, and was ableto suppress the signal from the tongue swab to 16% of the control tongueswab signal. The fact that false positive signals from tongue swabspecimens can be efficiently suppressed by addition of NAG suggests thatthe affinity purified Strep A antibody contains some non-specificbinding activities to human tissue or cells from the tongue/mouth.

Example 2: Dose Responses of NAG on Positive and False Positive Signals

Six tongue swabs were taken from a healthy individual. A Strep A qualitycontrol (QC) standard with 2.0×10³ organisms/test was obtained. Theswabs and the QC standards were prepared for testing using animmunoassay device for detection of Strep A using polyclonal Strep Aantibodies with a fluorescent label. Each sample was admixed with areagent comprising NAG at 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mLand 2 mg/mL. The samples were deposited on the immunoassay device, andthe test line was visually observed using a fluorescence analyzer forthe presence of a positive signal. Results are shown in FIG. 6 as afunction of concentration of NAG present in the extraction reagent fortongue swab samples (diamonds) and for a Strep A quality controlstandard of 2×10³ organisms/test (squares).

Example 3: Immunoassay Comprising NAG in Extraction Reagents

Thirty throat swabs collected from healthy donors were tested on animmunoassay for Strep A wherein NAG was added to the extraction mixtureat a concentration of 0.5 mg/mL. The extracted samples were deposited onan immunoassay, and the test line was viewed using a fluorescenceanalyzer to obtain an absolute count of signal emitted. An aliquot ofthe sample from each patient was also tested without using NAG in theextraction reagent (data in FIG. 1, and represented in FIG. 7 forcomparison). Results are shown in FIG. 7, where the bar on the righthand side of each data set for each patient corresponds to the sampleprepared in the presence of NAG. The results show that some throat swabspecimens from healthy donors can result in a strong false positivesignal, but these false positives can be suppressed dramatically by 0.5mg/mL NAG. See specimen #18 (“C-018”), in particular. Specimen #24 was(“C-024”) confirmed to be Strep A positive by cell culture.

Example 4: Immunoassay Comprising NAG in Sample Pad of ImmunoassayDevice

Immunoassay devices were prepared with a known quantity of NAG in thesample pad. The devices were prepared by depositing to the sample pad,and allowing to dry, a solution of NAG at concentrations of 0.4 mg/mL,0.6 mg/mL, 0.8 mg/mL and 1 mg/mL.

Tongue swab samples and Strep A QC standards, along with a salinenegative control, were each tested on an immunoassay device with eachconcentration of NAG. Results are shown in FIG. 8, where the absolutesignal at the test line of a Strep A immunoassay device comprising NAGin the sample pad upon application of saline (negative control) is shownin the left bar of each data set, the quality control standard of 2×10³organisms/test is shown in the middle bar of each data set and thetongue swab is shown in the right bar of each data set.

Example 5: Specificity and Sensitivity of Strep A Immunoassay TestStrips Comprising NAG in Sample Pad

Kits comprising an immunoassay test strip for detection of Strep A wereprepared. The kits comprised the immunoassay test strip housed in acassette with a sample receiving chamber, a vial with a reagent solutionand a dropper tip that fits securely on the open end of the vial, asterile rayon swab, a positive control swab that is coated withheat-inactivated, non-infectious Group A Streptoccus, a negative controlswab that is coated with heat-inactivated, non-infectious Group CStreptoccus, and instructions for use. The immunoassay test stripcomprised NAG on the sample pad of the test strip and was designed witha fluorescent label readable by an instrument.

The test kits were used in a clinical study where two throat swabs wereobtained from 533 patients with symptoms suggestive of bacterialpharyngitis. One throat swab was transported on cold ice packs to acentral Reference Laboratory, streaked on a sheep blood agar plate (SBA)and cultured for up to 48 hours Immediately after streaking, this sameswab was tested in the fluorescent immunoassay test strip. Theperformance of the fluorescent immunoassay test strip was determined bycomparison of its result to the corresponding culture result. Bacterialcultures with 10 or more Group A streptococcus (GAS)-positive coloniesin the first quadrant of the streak plate, and zero or more in the otherthree quadrants were considered culture-positive. The results from thisanalysis are presented in Table 6-1. SBA plates showing rare colonies,i.e. less than 10 colonies in the first quadrant and no growth in theother quadrants, were not included.

TABLE 6-1 Fluorescent Immunoassay results and Culture plate results fromsame swab SBA Culture: SBA Culture: Positive Negative Immunoassay 70 16test: Positive Immunoassay 0 432 test: Negative Total 70 448

From this data, the sensitivity is 70/70 (100%) (95% confidence interval(C.I.) 94-100%); specificity is 432/448 (96%) (95% C.I. 94-98%); thepositive predictive value is 81% and the negative predictive value is100%.

The distribution of GAS-positive cultures based on levels of bacterialgrowth on SBA plates and the corresponding results obtained with thefluorescent immunoassay test strip are presented in Table 6-2.Classification of culture results was also determined. Classificationwas based on the number of GAS positive colonies in each quadrant of thestreaked plate and ranged from rare (less than 10 colonies in the firstquadrant and no growth in the other quadrants) to 4+ (greater than 10colonies in all four quadrants). The fluorescent immunoassay test stripresults based on this culture classification are presented in Table 6-2.

TABLE 6-2 Culture Classification of Throat Swab Specimens andCorresponding Sofia Strep A FIA Results Fluorescent Immunoassay CultureClassification Test Strip Result Rare 10/15 (67%)  1+  9/9 (100%) 2+19/19 (100%) 3+ 25/25 (100%) 4+ 17/17 (100%)

The other throat swab, collected from the same patient, was testeddirectly in the physician's office or clinic without streaking on SBA.The results were compared to culture obtained with the other swab (seeTable 6-1 above). The sensitivity and specificity obtained with directtesting of this swab were 99% (69/70) and 96% (426/442), respectively.There were 15 rares (see Table 6-2 above) and six invalids; these wereexcluded from the calculations of clinical accuracy. The fluorescentimmunoassay test strip was also used to confirm the identification ofpresumptive Group A Streptococcus colonies on sheep blood agar plates.For culture confirmation, the test was 100% sensitive and 95% specific(Table 6-3).

TABLE 6-3 Confirmation of Bacterial Culture Results with Sofia Strep AFIA SBA Culture: SBA Culture: Positive Negative Immunoassay 17 1 test:Positive Immunoassay 0 20 test: Negative Total 17 21

From this data, the sensitivity is 17/17 (100%) (95% C.I. 78-100%\);specificity is 20/21 (96%) (95% C.I. 76-100%)); the positive predictivevalue is 94% and the negative predictive value is 100%.

While various specific embodiments have been illustrated and described,skilled artisans will recognize various modifications, permutations,additions and sub-combinations thereof, and will appreciate that thesecan be made without departing from the spirit and scope of the presentdisclosure. Therefore, it is to be understood that the disclosure is notto be limited to the specific embodiments disclosed herein, as such arepresented by way of example. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

All literature and similar materials cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, internet web pages and other publications cited in thepresent disclosure, regardless of the format of such literature andsimilar materials, are expressly incorporated by reference in theirentirety for any purpose to the same extent as if each were individuallyindicated to be incorporated by reference. In the event that one or moreof the incorporated literature and similar materials differs from orcontradicts the present disclosure, including, but not limited todefined terms, term usage, described techniques, or the like, thepresent disclosure controls.

What is claimed is:
 1. A device, comprising: a matrix comprising (i) asample receiving zone, (ii) a labeling zone comprising a labeledantibody with specific binding to Group A streptococcus antigen, to forma labeled antibody-bound antigen, and (iii) a capture zone comprising acapture antibody that binds the labeled antibody-bound antigen, whereinthe sample receiving zone, the labeling zone and the capture zone arearranged on the matrix in a liquid flow path, and wherein at least oneof the sample receiving zone and the labeling zone compriseN-acetyl-D-glucosamine (NAG) monomer to reduce the rate of falsepositives associated with detection of Group A streptococcus infection.2. The device of claim 1, wherein the labeled antibody with specificbinding to Group A streptococcus antigen is a polyclonal antibody. 3.The device of claim 2, wherein the polyclonal antibody is fluorescentlylabeled.
 4. The device of claim 1, wherein the labeled antibody withspecific binding to Group A streptococcus antigen is a monoclonalantibody.
 5. The device of claim 4, wherein the monoclonal antibody isfluorescently labeled.
 6. The device of claim 1, wherein the captureantibody is a polyclonal antibody.
 7. The device of claim 1, wherein thecapture antibody is a monoclonal antibody.
 8. A device, comprising: amatrix comprising (i) a sample receiving zone, (ii) a labeling zonecomprising a labeled antibody with specific binding to Group Astreptococcus antigen, to form a labeled antibody-bound antigen, and(iii) a capture zone comprising a capture antibody that binds thelabeled antibody-bound antigen, wherein the sample receiving zone, thelabeling zone and the capture zone are arranged on the matrix in aliquid flow path, and wherein at least one of the sample receiving zoneand the labeling zone comprise a reagent that blocks binding of thelabeled antibody with specific binding to Group A streptococcus antigenwith human epithelial cell wall glycoproteins, to reduce the rate offalse positives associated with detection of Group A streptococcusinfection.
 9. The device of claim 8, wherein the labeled antibody withspecific binding to Group A streptococcus antigen is a polyclonalantibody.
 10. The device of claim 9, wherein the polyclonal antibody isfluorescently labeled.
 11. The device of claim 8, wherein the labeledantibody with specific binding to Group A streptococcus antigen is amonoclonal antibody.
 12. The device of claim 11, wherein the monoclonalantibody is fluorescently labeled.
 13. The device of claim 8, whereinthe capture antibody is a polyclonal antibody.
 14. The device of claim8, wherein the capture antibody is a monoclonal antibody.
 15. The deviceof claim 8, wherein the reagent is selected from the group consisting ofN-acetyl glucosamine (NAG), glucosamine, acetyl-galactosamine,galactosamine, mannosamine, acetyl-muramic acid, chitin, chitosan andhyaluronic acid.
 16. A method for detecting the presence or absence ofGroup A streptococcus in a biological sample, comprising: providing adevice according to claim 8; placing a biological sample on the device;and determining the presence or absence of Group A streptococcus. 17.The method of claim 16, further comprising: providing an instrument forcollecting the biological sample; and collecting a biological sample onthe instrument.
 18. The method of claim 16, further comprising:providing instructions for use, wherein the instructions do not cautionto not touch the tongue, sides or top of mouth with the instrument whencollecting the sample.
 19. A kit, comprising: a device according toclaim 1; a container comprising an extraction reagent; an instrument forcollecting a biological sample; and instructions for use.
 20. The kit ofclaim 19, wherein the instrument is a swab.